JP6046144B2 - SRM / MRM assay for fatty acid synthase protein - Google Patents

Description

Background of the Invention

  This specification claims the benefit of US Provisional Application No. 61 / 538,091 (filed Sep. 22, 2011) entitled “SRM / MRM Assay for Fatty Acid Synthase Protein”, the entire contents of which are hereby incorporated by reference. Incorporated into.

Introduction Specific peptides are provided that are derived from subsequences of fatty acid synthase proteins (referred to herein as FASN, also referred to as FAS). Peptide sequences and fragmentation / transition ions for each peptide are particularly useful in mass spectrometry-based selective reaction monitoring (SRM) assays, which are also referred to as multiple reaction monitoring (MRM) assays. Such an assay is referred to herein as SRM / MRM. The use of peptides for quantitative analysis of FASN protein by SRM / MRM is described.

  This SRM / MRM assay can be used for the determination of the relative or absolute quantitative level of one or more specific peptides from the FASN protein. This provides a means of measuring the amount of FASN protein in a given protein preparation obtained from a biological sample by mass spectrometry.

More specifically, SRM / MRM assays can directly measure these peptides in complex protein lysates prepared from cells obtained from patient tissue samples such as formalin-fixed cancer patient tissue. Methods for preparing protein samples from formalin fixed tissue are described in US Pat. No. 7,473,532, the entire contents of which are hereby incorporated by reference. The method described in US Pat. No. 7,473,532 can be conveniently performed using Liquid Tissue ^™ reagent and protocols available from OncoPlexDx (formerly Expression Pathology Inc., Rockville, Md.).

  The most widely and advantageously available tissue forms from cancer patient tissues are formalin-fixed, paraffin-embedded tissues. Formaldehyde / formalin fixation of surgically removed tissue is by far the most common method for storing cancer tissue samples and is an accepted practice of standard pathological techniques. An aqueous solution of formaldehyde is called formalin. “100%” formalin consists of a saturated aqueous solution of formaldehyde (which is about 40% by volume or 37% by weight) and a small amount of stabilizer (usually methanol to limit the degree of oxidation and polymerization). The most common method of storing tissue is to immerse the entire tissue in an aqueous formaldehyde solution (commonly referred to as 10% neutral buffered formalin) for a long time (8-48 hours) and then to the entire fixed tissue Is embedded in paraffin wax for long-term storage at room temperature. Thus, molecular analysis methods for analyzing formalin-fixed cancer tissue will be the most acceptable and widely used method for analysis of cancer patient tissue.

  SRM / MRM assay results correlate accurate and precise quantification of FASN protein in specific tissue samples (eg, one or more cancer tissue samples) of patients or subjects from which tissues (biological samples) have been collected and stored Can be used for. This not only provides diagnostic information about the cancer, but also allows a doctor or other medical professional to determine the appropriate treatment for the patient. For example, such an assay can be designed to diagnose the stage or extent of cancer and determine the therapeutic agent most likely to respond to the patient. Such assays that provide diagnostic and therapeutically important information about protein expression levels in diseased tissue or other patient samples are called companion diagnostic assays.

  The assays described herein measure the relative or absolute level of unmodified specific peptides from the FASN protein and can also measure the absolute or relative level of modified specific peptides from the FASN protein. Examples of modifications include phosphorylated amino acid residues (eg, phosphotyrosine, phosphoserine and phosphothreonine) and glycosylated amino acid residues (eg, glycosylated asparagine residues) present in the peptide.

  The relative quantification level of FASN protein is determined by SRM / MRM techniques, eg, comparing SRM / MRM characteristic peak areas (eg, characteristic peak area or integrated fragment ionic strength) of individual FASN peptides in different samples. The Or, if each peptide has its own specific SRM / MRM feature peak, compare multiple SRM / MRM feature peak areas for multiple FASN feature peptides in one or more additional or different biological samples With FASN protein content, it is possible to determine the relative FASN protein content in one biological sample. Thus, the amount of a particular peptide from the FASN protein, and thus the amount of FASN protein, is determined relative to the same FASN peptide across two or more biological samples under the same experimental conditions. In addition, with the SRM / MRM technique, the feature peak area of the peptide can be compared with the feature peak area of another different peptide from a different protein in the same protein preparation from the same biological sample. Relative quantification can be determined for a given peptide from FASN protein. Thus, the amount of a particular peptide from the FASN protein, and thus the amount of FASN protein, is determined from each other within the same sample. These approaches result in quantification of individual peptides from the FASN protein to another peptide between samples and within the sample, where the absolute weight of the protein preparation from the biological sample relative to the volume of the FASN peptide or the weight relative to the weight. Regardless, the quantities determined by the peak areas are relative to each other. Relative quantitative data for individual feature peak areas between different samples is normalized to the amount of protein analyzed per sample. Relative quantification can be performed simultaneously over a single sample and / or many samples, over many peptides from multiple proteins and FASN proteins, with respect to the amount of protein relative to one peptide / protein relative to other peptides / proteins. Gain insight.

The absolute quantification level of the FASN protein is, for example, the SRM / MRM characteristic peak area of individual peptides from the FASN protein in one biological sample, and the SRM / MRM characteristic peak area of the “contaminated” internal standard added from the outside. Determined by the SRM / MRM method to be compared. In one embodiment, the internal standard is a synthetic version of the exact same FASN peptide comprising one or more amino acid residues labeled with one or more heavy isotopes. Appropriate isotope-labeled internal standards are predictable and consistent SRM / MRM features that, when analyzed by mass spectrometry, can be used as control peaks because each standard is different and distinguishable from the native FASN peptide feature peak Synthesized to produce a peak. Thus, when a known amount of an internal standard is added to a protein preparation from a biological sample and analyzed by mass spectrometry, the SRM / MRM characteristic peak area of the natural peptide from that sample is calculated as the SRM / MRM of the internal standard peptide. It can be compared with the characteristic peak area. This numerical comparison provides either the absolute molar concentration and / or the absolute weight of the natural peptide present in the initial protein preparation from the biological sample. The absolute quantification data for the fragment peptides is shown according to the amount of protein analyzed per sample. Absolute quantification can be performed simultaneously across many peptides, and thus proteins, across a single sample and / or many samples, providing insight into the absolute protein content of individual biological samples and the entire cohort of individual samples .
SRM / MRM assays are useful for directly diagnosing the stage of cancer in a patient-derived tissue such as, for example, formalin-fixed tissue, and determining the most advantageous therapeutic agent for use in treating the patient. Can be used to help. Analyze cancer tissue removed from the patient, either by surgery, such as the therapeutic removal of a part or whole of the tumor, or by biopsy methods performed to determine the presence or absence of a suspected disease, specific proteins Is present in the patient tissue and what form of protein is present. In addition, the expression level of the protein or proteins can be determined and compared to “normal” or reference levels found in healthy tissue. The normal or reference level of protein found in healthy tissue can be derived, for example, from the relevant tissue of one or more people who do not have cancer. Alternatively, the normal or reference level of a person with cancer can be obtained by analysis of related tissues that are not affected by the cancer.

  Protein level (eg, FASN level) assays can also be used to diagnose the stage of cancer in a patient or subject diagnosed with cancer using FASN levels. The level or amount of protein or peptide is defined as the quantity expressed in moles, which is the mass or weight of the protein or peptide as determined by the SRM / MRM assay. The level or amount can be normalized to the total level or amount of protein or another component in the lysate analyzed (eg, expressed in micromoles / micrograms of protein or micrograms of protein). Furthermore, the level or amount of protein or peptide can be determined on a volume basis, for example expressed in micromolar or nanogram / microliter. The level or amount of protein or peptide determined in the SRM / MRM assay can also be normalized to the number of cells analyzed. Thus, information about FASN can be used to determine the stage or grade of cancer by correlating the level of FASN protein (or fragment peptide of FASN protein) with the level found in normal tissues.

  Once the cancer stage and / or grade and / or FASN protein expression characteristics have been determined, the information can be used to identify cancer tissue characterized by abnormal expression of the analyzed protein (eg, FASN), for example. It can be adapted to the list of therapeutic agents (chemical and biological) developed to treat. Matching information in the FASN protein assay and a list of therapeutic agents that specifically target the FASN protein or cells / tissues that express the protein, for example, defines what is called a tailored medical approach to disease treatment. The assay methods described herein form the basis for a tailored medical approach by using analysis of proteins from the patient's own tissue as a source of diagnostic and therapeutic decisions.

Detailed description of the invention

  In principle, any predictive peptide derived from a FASN protein prepared, for example, by digestion with a protease of known specificity (eg, trypsin) can be used to determine the presence of FASN protein in a sample by mass spectrometry based SRM / MRM It can be used as a surrogate reporter for determination using the assay. Similarly, any predicted peptide sequence that contains an amino acid residue at a site known to be potentially modified in the FASN protein can also be used to analyze the extent of modification of the FASN protein in the sample. Yes.

FASN fragment peptides can be generated in a variety of ways including, for example, using the Liquid Tissue ^™ protocol described in US Pat. No. 7,473,532. The Liquid Tissue ^™ protocol and reagents generate peptide samples suitable for mass spectrometry from formalin-fixed paraffin-embedded tissue by protein digestion of tissue / biological samples. Suitable reagents and protocols are also commercially available from OncoPlexDx (formerly Expression Pathology Inc., Rockville, MD).

In the Liquid Tissue ^™ protocol, tissue / biological samples are heated in buffer for an extended period of time (eg, about 80 ° C. to about 100 ° C. for about 10 minutes to about 4 hours) to reverse or break protein cross-linking. The The buffer used is a neutral buffer (eg, a Tris base buffer or a buffer containing a detergent). Following heat treatment, the tissue / biological sample disrupts the tissue and cellular structure of said biological sample with one or more proteases including but not limited to trypsin, chymotrypsin, pepsin, and endoproteinase Lys-C, It is processed for a time sufficient to liquefy the sample. Exemplary conditions for protease treatment are 30 minutes to 24 hours at a temperature of 37 ° C to 65 ° C. Preferably, endoproteases used either simultaneously or sequentially, and in particular a combination of two or three endoproteases, are used to liquefy the sample. For example, suitable combinations of proteases can include, but are not limited to, combinations of trypsin, endoproteinase Lys-C and chymotrypsin, such as trypsin and endoproteinase Lys-C. The result of heating and protein digestion is a liquid, soluble dilutable biomolecule lysate. Advantageously, this liquid lysate does not have a solid or particulate material that can be separated from the lysate by centrifugation.

Surprisingly, many potential peptide sequences from FASN proteins have proved inappropriate or ineffective for use in mass spectrometry-based SRM / MRM assays for reasons that are not readily apparent. Since it was not possible to predict the most suitable peptide for the MRM / SRM assay, experimentally identified modified and unmodified peptides in actual Liquid Tissue ^TM lysates were used to ensure that the FASN protein There was a need to develop an accurate SRM / MRM assay. Without being bound by any theory, some peptides may be detected by mass spectrometry, for example, because they do not ionize well or do not produce fragments that differ from those produced from other proteins. It is thought that it may be difficult. Peptides may not resolve well in separation (eg, liquid chromatography) or may adhere to glass or plastic containers, leading to incorrect results in SRM / MRM assays.

The FASN peptides found in various embodiments of the present disclosure (eg, Tables 1 and 2 below) are available for all proteins in complex Liquid Tissue ^™ lysates prepared from cells sourced from formalin-fixed cancer tissue. Derived from FASN protein by protease digestion. Unless otherwise noted, the protease in each example was trypsin. Liquid Tissue ^™ lysates were then analyzed by mass spectrometry to determine those peptides derived from FASN proteins that were detected and analyzed by mass spectrometry. Certain specific subset of preferred peptides for mass spectrometry, 1) which peptides from proteins experimental determination of whether ionizing mass spectrometry of Liquid Tissue ^TM lysate and 2) Liquid Tissue ^TM lysate Based on the protocol used for the preparation and the ability of the peptide to survive the experimental conditions. This latter property extends not only to the amino acid sequence of the peptide, but also to the ability of the modified amino acid residues in the peptide to survive in a modified form during sample preparation.

Protein lysates from cells generated directly from formalin (formaldehyde) fixed tissue were prepared using Liquid Tissue ^™ reagents and protocols. This involves taking the cells into a test tube by microdissection of the tissue and then heating the cells for a long time in Liquid Tissue ^™ buffer. If formalin-induced cross-linking is adversely affected, the tissue / cell is then digested to the end in a predictable manner using a protease such as trypsin. One skilled in the art will appreciate that other proteases, particularly endoproteases, can be used in place of or in addition to trypsin. Each protein lysate was used to prepare a group of peptides by digestion of an intact polypeptide using a protease or combination of proteases. Each Liquid Tissue ^TM lysate can be analyzed (eg, by ion trap mass spectrometry) to perform multiple global proteomic investigations of the peptide and identified by mass spectrometry of all cellular proteins in each protein lysate Data presented as identification of as many peptides as possible. An ion trap mass spectrometer or another form of mass spectrometer that can perform global profiling for the identification of as many peptides as possible from a single complex protein / peptide lysate may be employed. However, an ion trap mass spectrometer can be the optimal type of mass spectrometer to perform overall profiling of peptides. Although SRM / MRM assays can be developed and performed on any type of mass spectrometer including MALDI, ion trap, or triple quadrupole, instrument platforms for SRM / MRM assays are often triplex. It is considered a quadrupole instrument platform.

Once a single mass analysis of a single lysate under the conditions used has identified as many peptides as possible, the list of identified peptides is then checked to determine the proteins detected in that lysate Used to be. This process was repeated for multiple Liquid Tissue ^™ lysates and ordered a very large list of peptides into a single data set. The resulting data set represents peptides that can be detected in the biological sample of the analyzed type (after protease digestion), in particular the Liquid Tissue ^TM lysate of the biological sample, and thus peptides for specific proteins such as, for example, the FASN protein. Including.

In one embodiment, FASN tryptic peptides identified as useful for determining the absolute or relative amount of FASN receptor include SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 8 or more, or 10 or more, each of which is listed in Table 1. Each of these peptides was detected by mass spectrometry of Liquid Tissue ^™ lysates prepared from formalin-fixed, paraffin-embedded tissues. Thus, each of the peptides in Table 1 or any combination of these peptides (eg, one or more, two or more, three or more, four or more, five or more of these peptides listed in Table 1 6 or more, 8 or more, or 10 or more, and combinations of peptides particularly found in Table 2), quantitative SRM of FASN proteins in human biological samples, including those directly to formalin-fixed patient tissues / Peptide candidates for use in MRM assays. Table 2 shows additional information for three of the peptides shown in Table 1.

FASN tryptic peptides listed in Table 1 include those detected from Liquid Tissue ^™ lysates of several different formalin-fixed tissues of different human organs including prostate, colon and breast. Each of these peptides is useful for quantitative SRM / MRM assays of FASN protein in formalin fixed tissue. Further data analysis of these experiments showed no preference for any specific peptide from any particular organ site. Thus, each of these peptides is suitable for performing SRM / MRM assays of FASN protein on Liquid Tissue ^™ lysates of any formalin-fixed tissue derived from any biological sample or any organ site of the body.

  In one embodiment, the peptides of Table 1 or any combination thereof (eg, one or more, two or more, three or more, four or more, five or more of the peptides listed in Table 1, 6 or more, 8 or more, or 10 or more, and especially combinations with peptides also found in Table 2) depending on mass spectrometry, including but not limited to immunological methods (eg Western blot or ELISA) Not analyzed by methods. Information regarding the amount of peptide (absolute or relative), regardless of how it was obtained, suggests the presence (diagnosis) of the cancer in the subject, determines the stage / grade / status of the cancer, It can be used in any of the methods described herein, including providing a prognosis or determining a therapeutic agent or treatment regimen for a subject / patient.

Embodiments of the present disclosure include compositions containing one or more, two or more, three or more, four or more, five or more, six or more, eight or more, ten or more of the peptides of Table 1. . In some embodiments, the composition comprises a peptide from Table 2. A composition comprising a peptide may comprise one or more, two or more, three or more, four or more, five or more, six or more, eight or more, or ten or more peptides that are isotopically labeled. Each of the peptides can be labeled with one or more isotopes independently selected from the group consisting of ¹⁸ O, ¹⁷ O, ³⁴ S, ¹⁵ N, ¹³ C, ² H or combinations thereof. A composition comprising a peptide from a FASN protein need not contain all of the peptides from that protein (eg, a full set of tryptic peptides), whether or not they are isotopically labeled. In some embodiments, the composition comprises one or more, two or more, three or more, four or more, five or more, six or more, eight or more, or ten or more peptides from FASN, and in particular Does not contain the peptides in Table 1 or Table 2. The composition comprising the peptide can be in the form of a dried or lyophilized material, a liquid (eg, aqueous) solution or suspension, an array, or a blot.

  One consideration for performing an SRM / MRM assay is the type of instrument that can be used to analyze the peptide. SRM / MRM assays can be developed and performed on any type of mass spectrometer, including MALDI, ion trap, or triple quadrupole, but the most advantageous instrument platform for SRM / MRM assays is often , Considered a triple quadrupole equipment platform. Its mass spectrometer type analyzes single isolated target peptides in very complex protein lysates that can consist of hundreds to millions of individual peptides from all proteins contained within the cell Therefore, it can be considered as the most suitable device.

  In order to perform the SRM / MRM assay for each peptide derived from the FASN protein most efficiently, it is desirable to use information in addition to the peptide sequence for analysis. The additional information is directed and directed to mass spectrometers (eg, triple quadrupole mass spectrometers), and specific target peptides are analyzed intensively in the right way, allowing the assay to be performed efficiently. To be.

  Additional information about target peptides in general and specific FASN peptides includes: peptide monoisotopic mass, its precursor charge state, precursor m / z value, m / z transition ions, and ions for each transition ion Can contain one or more of the types. By way of example, for the three FASN peptides listed in Table 1, additional peptide information that can be used to develop an SRM / MRM assay for FASN protein is shown in Table 2. Similar additional information described for these three FASN peptides, shown in Table 2 as examples, can be applied to the preparation, acquisition, and analysis of other peptides included in Table 1.

  The following methods are: 1) to identify candidate peptides from the FASN protein that can be used in mass spectrometry-based SRM / MRM assays for FASN protein; 2) separate SRM / MRM assays or multiple assays for target peptides from FASN protein And 3) used quantitative analysis to apply cancer diagnosis and / or selection of optimal treatment.

Assay method
1. Identification of SRM / MRM candidate fragment peptides for FASN protein:
To digest the protein, prepare a Liquid Tissue ^™ protein lysate from a formalin-fixed biological sample using a protease (which may or may not contain trypsin).
b. ^Analyze all protein fragments of the Liquid Tissue ^TM lysate with an ion trap tandem mass spectrometer to identify all fragment peptides from the FASN protein, where individual fragment peptides are phosphorylated or glycosylated, etc. Without any peptide modifications.
c. ^Analyze all protein fragments of the Liquid Tissue ^™ lysate with an ion trap tandem mass spectrometer to identify all fragment peptides from FASN proteins with peptide modifications such as phosphorylated or glycosylated residues.
d. Although all peptides produced by a specific digestion method may be measured from the entire full-length FASN protein, preferred peptides used in the development of SRM / MRM assays were prepared from formalin-fixed biological samples Identified by mass spectrometry directly in complex Liquid Tissue ^™ protein lysates.
e. Peptides that are specifically modified (phosphorylated, glycosylated, etc.) in patient tissue and ionized in the mass spectrometer when analyzing Liquid Tissue ^™ lysates from formalin-fixed biological samples, and therefore detected Identified as candidate peptides for the assay of peptide modification of FASN protein.

2. Mass spectrometry assay for fragmented peptides from FASN protein
a. A SRM / MRM assay on a triple quadrupole mass spectrometer for individual fragment peptides identified in Liquid Tissue ^™ lysate is applied to peptides from FASN protein.
i. For optimal chromatographic conditions including but not limited to gel electrophoresis, liquid chromatography, capillary electrophoresis, nano reversed phase liquid chromatography, high performance liquid chromatography, or reverse phase high performance liquid chromatography Determine the optimal retention time of the fragment peptide.
ii. To develop an SRM / MRM assay for each peptide, the monoisotopic mass of the peptide, the charge state of each peptide precursor, the m / z value of each peptide precursor, the m / z transition ion of each peptide , And the ionic type of each transition ion of each fragment peptide.
iii. The SRM / MRM assay can then be performed on a triple quadrupole mass spectrometer using information from (i) and (ii), where each peptide is in triple quadrupole mass spectrometry. When run in total, it has features that accurately define a unique SRM / MRM assay and a unique SRM / MRM feature peak.
b. The amount of FASN protein fragment peptide detected as a function of the unique SRM / MRM feature peak area from SRM / MRM mass spectrometry indicates both the relative and absolute amount of protein in a particular protein lysate Conduct SRM / MRM analysis so that you can.
i. Relative quantification can be achieved by:
1. SRM / MRM characteristic peak area from a given FASN peptide detected in Liquid Tissue ^™ lysate from one formalin-fixed biological sample, at least a second, third, fourth or more formalin-fixed biological at least a second from the sample, the third, by comparing the same SRM / MRM features peak area of the fourth or more Liquid Tissue ^TM same FASN fragment peptides in the lysate, an increase or decrease in the presence of FASN protein decide.
2. SRM / MRM characteristic peak areas from a given FASN peptide detected in Liquid Tissue ^TM lysate from one formalin-fixed biological sample, and other protein fragments from other samples from different biological sources Determine the increase or decrease in the presence of FASN protein by comparing to the SRM / MRM feature peak area generated from the peptide, where the comparison of the SRM / MRM feature peak area between the two samples for the peptide fragment is Normalized to the amount of protein analyzed in each sample.
3. To normalize the changing level of FASN protein relative to other proteins whose expression levels do not change under various cellular conditions, the SRM / MRM characteristic peak area for a given FASN peptide is calculated using the formalin-fixed biological sample. Increase or decrease in the presence of FASN protein is determined by comparison with SRM / MRM feature peak areas from other fragment peptides from different proteins within the same Liquid Tissue ^™ lysate.
4. These assays are applicable to both unmodified and modified fragment peptides of FASN protein, including but not limited to phosphorylation and / or glycosylation, relative levels of modified peptides Is determined in the same way as the relative amount of unmodified peptide is determined.
ii. Absolute quantification of a given peptide is based on the SRM / MRM characteristic peak area for a given fragment peptide from the FASN protein of an individual biological sample, and the SRM of the fragment peptide internal standard added to the protein lysate from that biological sample. / MRM feature peak area can be achieved by comparison.
1. The internal standard is a labeled synthetic version of the fragment peptide from the FASN protein under investigation. This standard mixes known amounts into the sample, and the SRM / MRM characteristic peak area can be determined separately for both the fragment peptide internal standard and the natural fragment peptide in the biological sample, and then both peak areas can be compared .
2. If the modification includes, but is not limited to, phosphorylation and / or glycosylation, and the absolute level of the modified peptide can be determined in the same way as determining the absolute level of the unmodified peptide, this is the unmodified fragment peptide And can be applied to modified fragment peptides.

3. Application of fragment peptide quantification to cancer diagnosis and treatment
a. Relative and / or absolute quantification of FASN protein fragment peptide levels, well known in the cancer field, between FASN protein expression and cancer stage / grade / status of patient tumor tissue Demonstrate that previously determined relevance is confirmed.
b. Perform relative and / or absolute quantification of fragment peptide levels of FASN protein to demonstrate correlation with clinical outcomes of different treatment strategies, but is this correlation already demonstrated in the field, Or it can be demonstrated in the future through correlation studies across patient cohorts and tissues from these patients. Once either a previously established correlation or a future occurring correlation is confirmed by the assay, the assay method can then be used to determine the optimal therapeutic strategy.

Assessment of FASN protein levels in tissues, based on analysis of formalin-fixed patient-derived tissues, can provide information related to diagnosis, prognosis, and treatment for each specific patient. In one embodiment, this disclosure describes a method for measuring FASN protein levels in a biological sample, including one or more modifications or unmodified proteins in a protein digest prepared from the biological sample. Detecting and / or quantifying the amount of modified FASN fragment peptide using mass spectrometry and calculating the level of modified or unmodified FASN protein in the sample, the level being relative or absolute Is a level. In a related embodiment, quantifying one or more FASN fragment peptides comprises determining the amount of each of the FASN fragment peptides in the biological sample by comparing to a known amount of an additional internal standard peptide, Each of the FASN fragment peptides in it is compared with an internal standard peptide having the same amino acid sequence. In some embodiments, the internal standard is an isotope-labeled internal standard comprising one or more stable heavy isotopes selected from ¹⁸ O, ¹⁷ O, ³⁴ S, ¹⁵ N, ¹³ C, ² H or combinations thereof. It is a peptide.

  The method for measuring the level of FASN protein in a biological sample (or a fragment peptide as a surrogate thereof) described herein can be used as a diagnostic indicator of cancer in a patient or subject. In one embodiment, the FASN protein level measurement is used to correlate the level of FASN protein found in tissue with that protein level found in normal and / or cancer or precancerous tissue (e.g., By comparing), the diagnostic stage / grade / status of cancer can be determined.

Embodiment:
1. A method for measuring fatty acid synthase (FASN) protein levels in a biological sample, wherein the amount of one or more modified or unmodified FASN fragment peptides in a protein digest prepared from the biological sample is determined by mass spectrometry. Using and detecting and / or quantifying, and calculating the level of modified or unmodified FASN protein in said sample, wherein said amount is a relative or absolute amount.
2. The method of embodiment 1, further comprising fractionating the protein digest prior to detecting and / or quantifying the amount of one or more modified or unmodified FASN fragment peptides.
3. The fractionation step is selected from the group consisting of gel electrophoresis, liquid chromatography, capillary electrophoresis, nano reverse phase liquid chromatography, high performance liquid chromatography, or reverse phase high performance liquid chromatography Form 2 method.
4. The method of any of embodiments 1-3, wherein the protein digest of the biological sample is prepared by the Liquid Tissue ^™ protocol.
5. The method of any of embodiments 1-3, wherein the protein digest comprises a protease digest.
6. The method of embodiment 5, wherein the protein digest comprises a trypsin digest.
7. Embodiments 1-6, wherein the mass spectrometry comprises tandem mass spectrometry, ion trap mass spectrometry, triple quadrupole mass spectrometry, MALDI-TOF mass spectrometry, MALDI mass spectrometry, and / or time-of-flight mass spectrometry Either way.
8. The method of embodiment 7, wherein the mode of mass spectrometry used is selective reaction monitoring (SRM), multiple reaction monitoring (MRM), and / or multiple selection reaction monitoring (mSRM), or any combination thereof.
9. One or more modified or unmodified FASN fragment peptides are SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 Comprising different amino acid sequences independently selected from those defined as SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11. The method of any one of forms 1-8.
10. The method of any of embodiments 1-9, wherein the biological sample is a blood sample, urine sample, serum sample, ascites sample, sputum sample, lymph, saliva sample, cell, or solid tissue.
11. The method of embodiment 10, wherein the tissue is formalin fixed tissue.
12. The method of embodiment 10 or 11, wherein the tissue is paraffin embedded tissue.
13. The method of embodiment 10, wherein the tissue is obtained from a tumor.
14. The method of embodiment 13, wherein the tumor is a primary tumor.
15. The method of embodiment 13, wherein the tumor is a secondary tumor.
16. The method of any of embodiments 1-15, further comprising quantifying the modified or unmodified FASN fragment peptide.
17. For quantification of FASN fragment peptide, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, comprising amino acid sequences of about 8 to about 45 amino acid residues of FASN shown as SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11 Including comparing the amount of one or more, two or more, three or more, four or more, or five or more FASN fragment peptides with the amount of the same FASN fragment peptide in different separate biological samples The method of form 16.
18. Quantifying one or more FASN fragment peptides comprises determining the amount of each FASN fragment peptide in a biological sample by comparing to a known amount of an additional internal standard peptide, comprising: Embodiment 18. The method of embodiment 17, wherein each of the peptides is compared to an internal standard peptide having the same amino acid sequence.
19. The method of embodiment 18, wherein the internal standard peptide is an isotope labeled peptide.
20. The embodiment wherein the isotope-labeled internal standard peptide comprises one or more stable heavy isotopes selected from ¹⁸ O, ¹⁷ O, ³⁴ S, ¹⁵ N, ¹³ C, ² H, or any combination thereof 19 ways.
21. Embodiments in which the detection and / or quantification of the amount of one or more modified or unmodified FASN fragment peptides in a protein digest indicates the presence of the modified or unmodified FASN protein and its association with cancer in a subject. Any method of 1-20.
22. Further comprising associating the result of the detection and / or quantification of the amount of one or more modified or unmodified FASN fragment peptides, or the amount of the FASN protein with the diagnostic stage / grade / status of the cancer The method of embodiment 21.
23. As a result of the detection and / or quantification of the amount of one or more modified or unmodified FASN fragment peptides, or associating the amount of the FASN protein with the diagnostic stage / grade / status of cancer, The method of embodiment 22, which is combined in multiple formats with detection and / or quantification of the amount of peptides from other proteins or other proteins to provide additional information about the diagnostic stage / grade / status of the cancer .
24. The method of Embodiments 1-23, further comprising selecting treatment based on the presence or amount of one or more FASN fragment peptides, or the amount of FASN protein for the subject from which the biological sample was obtained Method.
25. further comprising administering a therapeutically effective amount of the therapeutic agent to the patient from whom the biological sample was obtained, wherein the amount of therapeutic agent and / or administered therapeutic agent is one or more modified or unmodified FASN fragment peptides The method of any one of embodiments 1-24, which is based on the amount of or the amount of FASN protein.
26. The method of embodiments 24 and 25, wherein the therapeutic or therapeutic agent is against cancer cells expressing FASN protein.
27. The embodiment of embodiments 1-26, wherein the biological sample is formalin-fixed tumor tissue that has been treated to quantify the amount of one or more modified or unmodified FASN fragment peptides using the Liquid Tissue ^™ protocol and reagents. Method.
28. The one or more modified or unmodified FASN fragment peptides are two or more, three or more, four or more, five or more, six or more, eight or more, or ten or more of the peptides of Table 1. The method of any of embodiments 1-27.
29. Embodiments 1--including quantifying the amount of one, two or more, three or more, four or more, five or more, six or more, eight or more, or ten or more of the peptides of Table 2 One of 28 methods.
30. A composition comprising one, two or more, three or more, four or more, five or more, six or more, eight or more, or ten or more of the peptides of Table 1 or antibodies thereof, said composition A composition that optionally excludes one, two, three or more other peptides of FASN.
31. The composition of embodiment 30, comprising one, two, or three of the peptides of Table 2 or antibodies thereof.

  It should be understood that the description, specific examples, embodiments, and data in providing exemplary aspects are provided for purposes of illustration and are not intended to limit the present disclosure. Various changes and modifications within this disclosure will become apparent to those skilled in the art from the discussion, detailed description, and data contained herein, and are therefore considered part of the subject matter of this specification.

Claims (13)

A method for measuring fatty acid synthase (FASN) protein levels in a human biological sample that is a formalin-fixed tissue , wherein the amount of the SEQ ID NO: 4 FASN fragment peptide in the protein digest of the biological sample is measured using mass spectrometry detecting Oyo Bijou amount is, and includes calculating the level of F ASN protein in said sample,
The method wherein the amount is a relative amount or an absolute amount. Wherein prior to detection of Oyo Bijou amount the amount of FASN fragment peptides, further comprising the step of fractionating said protein digest method of claim 1. The fractionation step is a liquid chromatography, Nano reversed-phase liquid chromatography, it is selected from the group consisting of high performance liquid chromatography or reverse-phase high performance liquid chromatography, The method of claim 2. Comprising said protein digests tryptic digest method according to any one of claims 1 to 3. The method according to any one of claims 1 to 4 , wherein the tissue is a paraffin-embedded tissue. The method according to any one of claims 1 to 5 , wherein the tissue is obtained from a tumor. 7. The quantification of the FASN fragment peptide comprises comparing the amount of the FASN fragment peptide in one biological sample with the amount of the same FASN fragment peptide in different separate biological samples . The method according to one item . By determination of the FASN fragment peptide, compared to the known amount of added internal standard peptide, and determining the amount of pre-Symbol F ASN fragment peptide in a biological sample, said FASN fragment peptide in the biological sample but compared to an internal standard peptide having the same amino acid sequence, the internal standard peptide which is an isotope labeled peptide, the method according to any one of claims 1 to 6. Detection and / or quantification of the amount of the FASN fragment peptide of said protein digest indicates the association between cancer in the presence and the subject of a modified or unmodified FASN protein, any one of the preceding claims The method described in 1. The FASN fragment peptide of the amount of the detected Oyo Bijou amount of results, or the amount of the FASN protein, further comprising providing information to associate with diagnostic Stage / grade / status of said cancer, The method of claim 9 . The FASN fragment peptide of the amount of the detected Oyo Bijou amount of result, or the information for associating the amount of the FASN protein and diagnostic Stage / grade / status of said cancer, other proteins or other, a detection and / or quantification of the information of the amount of peptides from proteins, by combining in multiplex format, to provide additional information about the diagnostic stage / grade / status of the cancer claim 9 Or the method according to 10 . The relative test subjects the biological sample is acquired, the FASN presence of a fragment peptide or its amount or to assist in selecting a treatment or therapeutic agent based on the amount of FASN protein, according to claim 1 to 11 The method as described in any one of . The method according to claim 12 , wherein the treatment or the therapeutic agent is for cancer cells expressing the FASN protein.